Figure 4.

Neoplastic cells that lack density-dependent Lamγ2 repression display EGFR/ERK hyperactivation. A: The EGFR/ERK and PI3K/AKT/mTOR kinase cascade signaling pathways and their downstream targets analyzed in this study. Arrows indicate activation; T-bars indicate inhibition. Dashed arrows indicate a less important role for S6K1 in activating these targets. Note that S6K1 must first phosphorylate S6 at ser(240/244) before either RSK or S6K1 can phosphorylate S6 at ser(235/236). Underlining indicates proteins that were analyzed for phosphorylation/activation by Western blotting. For some experiments, some proteins (shown in blue) were expressed or hyperactivated by engineering normal keratinocytes. Inhibitors that specifically block the associated kinase are shown in red. B: Confluent cultures of normal (G5Ep, OKF6, strain N) and premalignant (N/E6E7, POE9n, D17) keratinocytes and SCC cells (SCC-13, SCC-71, SCC-15) analyzed by Western blotting for Lamγ2 and selected phosphoproteins. Note consistent p-EGFR, p-ERK, and p-S6 increases in all of the Lamγ2-overexpressing cell lines. C: Effects of kinase inhibitors on Lamγ2 and Lamβ3 expression and on signaling molecules. Confluent cultures of SCC-68, SCC-13, POE9n, and N/E6E7 and preconfluent cultures of G5Ep were treated for 2 days with kinase inhibitors and then were analyzed by Western blotting. TRI is the TGFβ receptor I inhibitor, gefitinib is an EGFR inhibitor, U0126 and AZD6244 are MEK inhibitors, and Ku-0063794 is a TORC1/2 inhibitor. Note reduction of Lamγ2 expression, but not of Lamβ3 expression, by all kinase inhibitors tested except Ku-0063794. Lamγ2 reduction correlated consistently with decreased p-ERK.